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Immunolocalization to Study ARGONAUTE Proteins in Developing Ovules of the Brassicaceae

  • Gloria León-Martínez
  • Edgar Demesa-Arévalo
  • Jean-Philippe Vielle-CalzadaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1932)

Abstract

Determining the in situ pattern of protein expression is crucial to accurately establish regulatory function and mode of action of any plant developmental program. Here, we describe two immunolocalization procedures that are consistently used to determine subcellular localization of ARGONAUTE proteins in the ovule of the Brassicaceae. The first is performed in resin-embedded semi-thin sections of developing ovules that can be observed under bright-field microscopy. The second is based in polyacrylamide immersion of complete (whole-mounted) gynoecia or ovules that are observed under confocal microscopy. Both procedures have been successfully performed to localize proteins involved in RNA-directed DNA methylation during the development of the anatropous bitegmic ovule in Arabidopsis, Brassica, or Boechera species.

Key words

ARGONAUTE Small RNAs Immunolocalization Female meiosis Ovule RdDM 

Notes

Acknowledgments

We are grateful to Rocio Escobar and Daniel Rodriguez for continuous efforts aiming at protocol improvement and to Lily Solorzano and Judith Lúa for help with reagent acquisition and purchasing. This research was supported by grants from the Consejo Nacional de Ciencia y Tecnología (CB2015/256826 and IFC2015-1-449).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Gloria León-Martínez
    • 1
  • Edgar Demesa-Arévalo
    • 1
  • Jean-Philippe Vielle-Calzada
    • 1
    Email author
  1. 1.Grupo de Desarrollo Reproductivo y ApomixisUnidad de Genómica Avanzada, Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN)IrapuatoMexico

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